Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders

ABSTRACT

The present invention relates to the prevention or treatment of skeletal growth retardation disorders, in particular skeletal diseases developed by patients that display abnormal increased activation of the fibroblast growth factor receptor 3 (FGFR3), in particular by expression of a constitutively activated mutant of FGFR3. More particularly, the present invention relates to a soluble FGFR3 for use in the prevention or treatment of achondroplasia.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/532,184 filed on Aug. 5, 2019 (now abandoned), which is acontinuation of U.S. application Ser. No. 14/759,490 filed on Jul. 7,2015 (now abandoned), which is a national stage filing under U.S.C. §371 of PCT International Application No. PCT/IB2013/001480, with aninternational filing date of Jan. 16, 2013, the contents of each ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the prevention or treatment of skeletalgrowth retardation disorders, in particular skeletal diseases andcraniosynostosis, developed by patients that display abnormal increasedactivation of the fibroblast growth factor receptor 3 (FGFR3), inparticular by expression of a constitutively activated mutant of FGFR3.

BACKGROUND OF THE INVENTION

Skeletal development in humans is regulated by numerous growth factors.

Among them Fibroblast Growth Factor Receptor 3 (FGFR3) has beendescribed as both a negative and a positive regulator of endochondralossification. Mutations in the gene encoding for the FGFR3 have beenshown to be responsible for the phenotype of numerous skeletalchondrodysplasias (1), including the thanatophoric dysplasias (TDI andTDII) (2) and achondroplasia (3), the most common form of short limbdwarfism. Children affected by achondroplasia suffer from deformationsof the skull and vertebrae and abnormal long bone development, resultingin short stature and severe neurological and orthopedic complications(4, 5). Existing treatments are only designed to alleviate some of thecomplications, and are invasive and extreme (6, 7)

Achondroplasia is an autosomal dominant disorder caused by a pointmutation in the gene for FGFR3 (Fgfr3ach) (8). In 97% of affectedpatients, achondroplasia is caused by a G380R substitution in thetransmembrane domain of the receptor (9, 10). This mutation in FGFR3results in a gain of function (11), which prolongs activation of thetyrosine kinase activity of the receptor (12, 13). The G380R mutantFGFR3 remains ligand dependent for its dimerization and activation (12,14); however, the presence of the mutation stabilizes theligand/receptor complex (15) and slows down receptor internalization(12), thus extending subsequent intracellular Ras/MAPK pathway signaling(12). The resultant FGFR3 signaling is prolonged and steadily inhibitschondrocyte proliferation and differentiation in the growth plate (16).Cells expressing the mutant receptor do not mature and are not replacedby mineralized bone matrix, impairing lengthening of all bones formed byendochondral ossification (17, 18). These include the long bones of theappendicular skeleton, as well as the vertebrae, sternum, cranial base,and some bones in the skull where bone growth occurs at synchondroses,which are cartilaginous structures consisting of two opposed growthplates with a common zone of resting chondrocytes. As with endochondralgrowth plates in the long bones, synchondroses also become replaced bybone.

Despite an increased number of studies deciphering the mechanismsresponsible for bone growth disturbances, there is still no cureavailable. Several therapeutic strategies are considered targetingmutant FGFR3 and its downstream signaling (16). Recently, Jin et al.have tested a novel peptide inhibiting FGFR3 signaling in a murine modelof TDII (19). This study shows reversion of the neonatal lethality ofTD11 mice following in utero treatment and demonstrates theproof-of-concept that targeting FGFR3 in the extracellular compartmentmay be an effective strategy to treat FGFR3-related skeletal dysplasias.

Current therapies of achondroplasia include orthopedic surgeries such asleg lengthening and growth hormone therapy. However, leg lengtheninginflicts a great pain on patients, and growth hormone therapy increasesbody height by means of periodic growth hormone injections starting fromchildhood. Further, growth ceases when injections are stopped.Consequently, it is desirable to develop a new achondroplasia therapy,as well as other skeletal growth retardation disorders includingFGFR3-related skeletal diseases.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an isolated solubleFibroblast Growth Factor Receptor 3 (sFGFR3) polypeptide or a functionalequivalent thereof for use in the prevention or treatment of a skeletalgrowth retardation disorder.

In a second aspect, the present invention also relates to apharmaceutical composition comprising an isolated sFGFR3 polypeptide ora functional equivalent thereof and a pharmaceutically acceptablecarrier.

In a third aspect, the present invention further relates to apharmaceutical composition for use in the prevention or treatment of askeletal growth retardation disorder FGFR3-related skeletal diseasecomprising an isolated sFGFR3 polypeptide or a functional equivalentthereof and a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a method forpreventing or treating a skeletal growth retardation disorderFGFR3-related skeletal disease comprising the step of administering atherapeutically effective amount of a sFGFR3 polypeptide or apharmaceutical composition comprising such polypeptide to a subject inneed thereof.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have designed an effective therapeutic approach forachondroplasia by restoring bone growth. As shown herein, post-nataladministration of recombinant soluble fibroblast growth factor receptor3 (sFGFR3) acting as a decoy receptor to Fgfr3^(ach/+) mice (a murinemodel of achondroplasia displaying a phenotype essentially identical tothe human pathology, with shortening of all bones formed by endochondralossification) results in normal skeletal growth preventing onset ofachondroplasia symptoms and complications.

As disclosed herein, repeated subcutaneous injections of recombinantsFGFR3 throughout the growth period, normal skeletal growth can berestored in transgenic Fgfr3^(ach/+) mice, resulting in normal bodylength and significant decrease in associated complications. Effectivematuration of growth plate chondrocytes was restored in bones of treatedmice, resulting in a dose-dependent enhancement of skeletal growth inFgfr3^(ach/+) mice. This resulted in normal stature associated withsignificant decrease in number and intensity of complications, withoutany evidence of toxicity. These results validate the use of solubleFGFR3 to restore bone growth and indicate its potential use as apromising therapy for achondroplasia and related skeletal disorders.

Therapeutic Methods and Uses

The present invention provides methods and compositions (such aspharmaceutical compositions) for preventing or treating a skeletalgrowth retardation disorder.

The present invention relates thus to an isolated soluble FibroblastGrowth Factor Receptor 3 (sFGFR3) polypeptide or a functional equivalentthereof for use in the prevention or treatment of a skeletal growthretardation disorder.

In one embodiment, the skeletal growth retardation disorder is anidiopathic skeletal growth retardation disorder.

In another embodiment, the skeletal growth retardation disorder is aFGFR3-related skeletal disease.

The terms “Fibroblast Growth Factor Receptor 3” (“FGFR3”) or “FGFR3receptor”, as used herein, refer to any native or variant FGFR3polypeptide. The FGFR3 gene, which is located on the distal short arm ofchromosome 4, encodes a 806 amino acid protein precursor (fibroblastgrowth factor receptor 3 isoform 1 precursor). The naturally occurringhuman FGFR3 gene has a nucleotide sequence as shown in Genbank Accessionnumber NM_000142.4 and the naturally occurring human FGFR3 protein hasan aminoacid sequence as shown in Genbank Accession number NP_000133.

The term “polypeptide” means herein a polymer of amino acids having nospecific length. Thus, peptides, oligopeptides and proteins are includedin the definition of “polypeptide” and these terms are usedinterchangeably throughout the specification, as well as in the claims.The term “polypeptide” does not exclude post-translational modificationsthat include but are not limited to phosphorylation, acetylation,glycosylation and the like.

By an “isolated” polypeptide, it is intended that the polypeptide is notpresent within a living organism, e.g. within human body. However, theisolated polypeptide may be part of a composition or a kit. The isolatedpolypeptide is preferably purified.

A “native sequence” polypeptide refers to a polypeptide having the sameamino acid sequence as a polypeptide derived from nature. Thus, a nativesequence polypeptide can have the amino acid sequence ofnaturally-occurring polypeptide from any mammal (including human. Suchnative sequence polypeptide can be isolated from nature or can beproduced by recombinant or synthetic means. The term “native sequence”polypeptide specifically encompasses naturally-occurring truncated orsecreted forms of the polypeptide (e. g., an extracellular domainsequence), naturally-occurring variant forms (e. g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide.

A polypeptide “variant” refers to a biologically active polypeptidehaving at least about 80% amino acid sequence identity with the nativesequence polypeptide. Such variants include, for instance, polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide. Ordinarily, a variant will have atleast about 80% amino acid sequence identity, more preferably at leastabout 90% amino acid sequence identity, and even more preferably atleast about 95% amino acid sequence identity with the native sequencepolypeptide.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

In the frame of the present application, the percentage of identity iscalculated using a global alignment (i.e., the two sequences arecompared over their entire length). Methods for comparing the identityand homology of two or more sequences are well known in the art. The“needle” program, which uses the Needleman-Wunsch global alignmentalgorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to findthe optimum alignment (including gaps) of two sequences when consideringtheir entire length, may for example be used. The needle program is forexample available on the ebi.ac.uk world wide web site. The percentageof identity in accordance with the invention is preferably calculatedusing the EMBOSS::needle (global) program with a “Gap Open” parameterequal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62matrix.

Polypeptides consisting of an amino acid sequence “at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. The polypeptide consisting of anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a reference sequence may correspond to an allelic variantof the reference sequence. It may for example only comprisesubstitutions compared to the reference sequence. The substitutionspreferably correspond to conservative substitutions as indicated in thetable below.

Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Met,Pro, Amino acids with aliphatic hydrophobic Phe, Trp side chains Ser,Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp,Glu Amino acids with acidic side chains Lys, Arg, His Amino acids withbasic side chains Gly Neutral side chain

A soluble FGFR3 polypeptide exerts an inhibitory effect on thebiological activity of the FGFs proteins by binding to these proteins,thereby preventing them from binding to FGFR3 present on the surface oftarget cells. It is undesirable for a soluble FGFR3 polypeptide not tobecome associated with the cell membrane. In a preferred embodiment, thesoluble FGFR3 polypeptide lacks any amino acid sequences correspondingto the transmembrane and/or intracellular domains from the FGFR3polypeptide from which it is derived.

The terms “soluble FGFR3 polypeptide” or “sFGFR3”, as used herein, referto a polypeptide comprising or consisting of the extracellular region ofthe FGFR3 or a fragment thereof. For example, sFGFR3 may include all theextracellular domain of human FGFR3 (i.e. a polypeptide comprising orconsisting of the amino acid sequence ranging from positions 1-694 ofhuman FGFR3 as shown by SEQ ID NO: 1 below).

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGINPSERNMVGPQRLQVINASHEDSGAYSCRQRUTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLIMQQWSLVMESVVPSDRGNYTCVVENKFGKRQTYTLDVILERSPHRPILQAGLPANQTAVLGSDVEFFICKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVIKVSLESNASMSSNTPLVRLARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVIVINEAIGIDKDRAAKTVTVAVKIALKDDATDKDLSDLVSEMEMMKMIGKHKNHNLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVIVIKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLUCEGFERMDKPANCTEIDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPSSGGSR T

In one particular embodiment, the sFGFR3 polypeptide is encoded by anucleic acid sequence defined by SEQ ID NO: 2 (below).

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCCTAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCGACATCCAGTGGCTCAAGCACGTGGAGGTGATTGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGCTGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATCGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACAACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTTCACTGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATGATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGACGACTCCGTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGCAGTGGGGGCTCGCGGACG

Such nucleic acid sequence has been optimized to decrease GC content(while encoding for the native polypeptide sequence) in order to prolongmRNA half life.

A “functional equivalent” is a molecule (e.g. a recombinant polypeptide)that retains the biological activity and the specificity of the parentpolypeptide. Therefore, the term “functional equivalent of sFGFR3”includes variants and fragments of the polypeptide to which it refers(i.e. the sFGFR3 polypeptide) provided that the functional equivalentsexhibit at least one, preferably all, of the biological activities ofthe reference polypeptide, for instance retains the capacity of bindingto the FGFs proteins. As used herein, “binding specifically” means thatthe biologically active fragment has high affinity for FGFs but not forcontrol proteins. Specific binding may be measured by a number oftechniques such as ELISA, flow cytometry, western blotting, orimmunoprecipitation. Preferably, the functionally equivalentspecifically binds to FGFs at nanomolar or picomolar levels.

Thus, the polypeptide according to the invention encompassespolypeptides comprising or consisting of fragments of the extracellularregion of the FGFR3, provided the fragments are biologically active. Inthe frame of the invention, the biologically active fragment may forexample comprise at least 300, 400, 500, 600 or 650 consecutive aminoacids of the extracellular region of the FGFR3 receptor.

By “biological activity” of a functional equivalent of the extracellularregion of the FGFR3 receptor is meant (i) the capacity to bind to FGFs;and/or (ii) the capacity to reduce FGF intracellular signaling (e.g. Erkphosphorylation following FGFR3 receptor activation by its binding withFGFs); and/or (iii) the capacity to restore bone growth in vivo (e.g. inFgfr3^(ach/+) mice).

S The skilled in the art can easily determine whether a functionalequivalent of the extracellular region of the FGFR3 is biologicallyactive. To check whether the newly generated polypeptides bind to FGFsand/or reduce FGF intracellular signaling in the same way than theinitially characterized polypeptide sFGFR3 (a polypeptide consisting ofthe sequence depicted in SEQ ID NO: 1) a binding assay, a FGF activityassay or an ERK Activation Assay (see in Example) may be performed witheach polypeptide. Additionally, a time-course and a dose-responseperformed in vitro or in vivo (e.g. by using Fgfr3^(ach/+) mice asdescribed in the Examples section) will determine the optimal conditionsfor each polypeptide.

Moreover, it should be further noted that functional activation of theFGFR3 receptor may be readily assessed by the one skilled in the artaccording to known methods. Indeed, since activated FGFR3 receptor isphosphorylated on tyrosine residues located towards the cytoplasmicdomain, i.e. on Tyr⁶⁴⁸ and Tyr⁶⁴⁷, functional activation of the FGFR3receptor may for example be assessed by measuring its phosphorylation.

For instance, analysis of ligand-induced phosphorylation of the FGFR3receptor can be performed as described in Le Corre et al. (Org. Biomol.Chem., 8: 2164-2173, 2010).

Alternatively, receptor phosphorylation in cells can be readily detectedby immunocytochemistry, immunohistochemistry and/or flow cytometry usingantibodies which specifically recognize this modification. For instancephosphorylation of FGFR3 on the Tyro⁶⁴⁸ and Tyr⁶⁴⁷ residues can bedetected by immunocytochemistry, immunohistochemistry and/or flowcytometry using monoclonal or polyclonal antibodies directed againstphosphorylated Tyr⁶⁴⁸ and Tyr⁶⁴⁷-FGFR3.

Further, FGFR3, when associated with its ligand, mediates signaling byactivating the ERK and p38 MAP kinase pathways, and the STAT pathway.Therefore activation of FGFR3 receptor can also be assessed bydetermining the activation of these specific pathways as described byHorton et al. (Lancet, 370: 162-172, 2007)

In one embodiment, the polypeptides of the invention may comprise a tag.A tag is an epitope-containing sequence which can be useful for thepurification of the polypeptides. It is attached to by a variety oftechniques such as affinity chromatography, for the localization of saidpeptide or polypeptide within a cell or a tissue sample usingimmunolabeling techniques, the detection of said polypeptide byimmunoblotting etc. Examples of tags commonly employed in the art arethe GST (glutathion-S-transferase)-tag, the FLAG™-tag, the Strep-tag™,V5 tag, myc tag. His tag (which typically consists of six histidineresidues), etc.

In another embodiment, the polypeptides of the invention may comprisechemical modifications improving their stability and/or theirbiodisponibility. Such chemical modifications aim at obtainingpolypeptides with increased protection of the polypeptides againstenzymatic degradation in vivo, and/or increased capacity to crossmembrane barriers, thus increasing its half-life and maintaining orimproving its biological activity. Any chemical modification known inthe art can be employed according to the present invention. Suchchemical modifications include but are not limited to:

-   -   replacement(s) of an amino acid with a modified and/or unusual        amino acid, e.g. a replacement of an amino acid with an unusual        amino acid like Nle, Nva or Orn; and/or    -   modifications to the N-terminal and/or C-terminal ends of the        peptides such as e.g. N-terminal acylation (preferably        acetylation) or desamination, or modification of the C-terminal        carboxyl group into an amide or an alcohol group;    -   modifications at the amide bond between two amino acids:        acylation (preferably acetylation) or alkylation (preferably        methylation) at the nitrogen atom or the alpha carbon of the        amide bond linking two amino acids;    -   modifications at the alpha carbon of the amide bond linking two        amino acids such as e.g. acylation (preferably acetylation) or        alkylation (preferably methylation) at the alpha carbon of the        amide bond linking two amino acids.    -   chirality changes such as e.g. replacement of one or more        naturally occurring amino acids (L enantiomer) with the        corresponding D-enantiomers;    -   retro-inversions in which one or more naturally-occurring amino        acids (L-enantiomer) are replaced with the corresponding        D-enantiomers, together with an inversion of the amino acid        chain (from the C-terminal end to the N-terminal end);    -   azapeptides, in which one or more alpha carbons are replaced        with nitrogen atoms; and/or    -   betapeptides, in which the amino group of one or more amino acid        is bonded to the β carbon rather than the α carbon.

In another embodiment, adding dipeptides can improve the penetration ofa circulating agent in the eye through the blood retinal barrier byusing endogenous transporters.

Another strategy for improving drug viability is the utilization ofwater-soluble polymers. Various water-soluble polymers have been shownto modify biodistribution, improve the mode of cellular uptake, changethe permeability through physiological barriers; and modify the rate ofclearance from the body. To achieve either a targeting orsustained-release effect, water-soluble polymers have been synthesizedthat contain drug moieties as terminal groups, as part of the backbone,or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, givenits high degree of biocompatibility and ease of modification. Attachmentto various drugs, proteins, and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chain and viaother chemical methods; however, PEG itself is limited to at most twoactive agents per molecule. In a different approach, copolymers of PEGand amino acids were explored as novel biomaterials which would retainthe biocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule (providing greaterdrug loading), and which could be synthetically designed to suit avariety of applications.

Those of skill in the art are aware of PEGylation techniques for theeffective modification of drugs. For example, drug delivery polymersthat consist of alternating polymers of PEG and tri-functional monomerssuch as lysine have been used by VectraMed (Plainsboro, N.J.). The PEGchains (typically 2000 daltons or loss) are linked to the a- and c-aminogroups of lysine through stable urethane linkages. Such copolymersretain the desirable properties of PEG, while providing reactive pendentgroups (the carboxylic acid groups of lysine) at strictly controlled andpredetermined intervals along the polymer chain. The reactive pendentgroups can be used for derivatization, cross-linking, or conjugationwith other molecules. These polymers are useful in producing stable,long-circulating pro-drugs by varying the molecular weight of thepolymer, the molecular weight of the PEG segments, and the cleavablelinkage between the drug and the polymer. The molecular weight of thePEG segments affects the spacing of the drug/linking group complex andthe amount of drug per molecular weight of conjugate (smaller PEGsegments provides greater drug loading). In general, increasing theoverall molecular weight of the block co-polymer conjugate will increasethe circulatory half-life of the conjugate. Nevertheless, the conjugatemust either be readily degradable or have a molecular weight below thethreshold-limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintainingcirculatory half-life, and biodistribution, linkers may be used tomaintain the therapeutic agent in a pro-drug form until released fromthe backbone polymer by a specific trigger, typically enzyme activity inthe targeted tissue. For example, this type of tissue activated drugdelivery is particularly useful where delivery to a specific site ofbiodistribution is required and the therapeutic agent is released at ornear the site of pathology. Linking group libraries for use in activateddrug delivery are known to those of skill in the art and may be based onenzyme kinetics, prevalence of active enzyme, and cleavage specificityof the selected disease-specific enzymes. Such linkers may be used inmodifying the protein or fragment of the protein described herein fortherapeutic delivery.

In still another embodiment, the polypeptides of the invention may befused to a heterologous polypeptide (i.e. polypeptide derived from anunrelated protein, for example, from an immunoglobulin protein).

As used herein, the terms “fused” and “fusion” are used interchangeably.These terms refer to the joining together of two more elements orcomponents, by whatever means including chemical conjugation orrecombinant means. An “in-frame fusion” refers to the joining of two ormore polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manncr that maintains the correct translational readingframe of the original ORFs. For instance, a recombinant fusion proteinmay be a single protein containing two or more segments that correspondto polypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence.

As used herein, the term “sFGFR3 fusion protein” refers to a polypeptidecomprising the FGFR3 polypeptide or a functional equivalent thereoffused to heterologous polypeptide. The FGFR3 fusion protein willgenerally share at least one biological property in common with theFGFR3 polypeptide (as described above).

An example of a sFGFR3 fusion protein is a sFGFR3 immunoadhesin.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The immunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain (Fc region). Immunoadhesins can possessmany of the valuable chemical and biological properties of humanantibodies. Since immunoadhesins can be constructed from a human proteinsequence with a desired specificity linked to an appropriate humanimmunoglobulin hinge and constant domain (Fc) sequence, the bindingspecificity of interest can be achieved using entirely human components.Such immunoadhesins are minimally immunogenic to the patient, and aresafe for chronic or repeated use. In one embodiment, the Fe region is anative sequence Fe region. In another embodiment, the Fe region is avariant Fc region. In still another embodiment, the Fc region is afunctional Fc region. The sFGFR3 portion and the immunoglobulin sequenceportion of the sFGFR3 immunoadhesin may be linked by a minimal linker.The immunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG1, IgG2, IgG3or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG1 or IgG3.

As used herein, the term “Fe region” is used to define a C-terminalregion of an immunoglobulin heavy chain, including native sequence Fcregions and variant Fe regions. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof.

Another example of a sFGFR3 fusion protein is a fusion of the sFGFR3polypeptide with human serum albumin-binding domain antibodies(AlbudAbs) according to the AlbudAb™ Technology Platform as described inKonterman et al. 2012 AlbudAb™ Technology Platform-Versatile AlbuminBinding Domains for the Development of Therapeutics with TunableHalf-Lives

The polypeptides of the invention may be produced by any suitable means,as will be apparent to those of skill in the art. In order to producesufficient amounts of a sFGFR3 or functional equivalents thereof, or asFGFR3 fusion protein such as a sFGFR3 immunoadhesin for use inaccordance with the invention, expression may conveniently be achievedby culturing under appropriate conditions recombinant host cellscontaining the polypeptide of the invention. Preferably, the polypeptideis produced by recombinant means, by expression from an encoding nucleicacid molecule. Systems for cloning and expression of a polypeptide in avariety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferablygenerated by expression from an encoding nucleic acid in a host cell.Any host cell may be used, depending upon the individual requirements ofa particular system. Suitable host cells include bacteria mammaliancells, plant cells, yeast and baculovirus systems. Mammalian cell linesavailable in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells. HeLa cells, baby hamster kidneycells and many others (e.g. HEK 293 cells). Bacteria are also preferredhosts for the production of recombinant protein, due to the ease withwhich bacteria may be manipulated and grown. A common, preferredbacterial host is E. coli.

Moreover, it should be noted that the majority of protein-basedbiopharmaceuticals bare some form of post-translational modificationwhich can profoundly affect protein properties relevant to theirtherapeutic application. Protein glycosylation represents the mostcommon modification (about 50% of human proteins are glycosylated).Glycosylation can introduce considerable heterogeneity into a proteincomposition through the generation of different glycan structures on theproteins within the composition. Such glycan structures are made by theaction of diverse enzymes of the glycosylation machinery as theglycoprotein transits the Endoplasmatic Reticulum (ER) and theGolgi-Complex (glycosylation cascade). The nature of the glycanstructure(s) of a protein has impact on the protein's folding,stability, life time, trafficking, pharmaco-dynamics, pharmacokineticsand immunogenicity. The glycan structure has great impact on theprotein's primary functional activity. Glycosylation can affect localprotein structure and may help to direct the folding of the polypeptidechain. One important kind of glycan structures are the so calledN-glycans. They are generated by covalent linkage of an oligosaccharideto the amino (N)-group of asparagin residues in the consensus sequenceNXS/T of the nascent polypeptide chain. N-glycans may furtherparticipate in the sorting or directing of a protein to its finaltarget: the N-glycan of an antibody, for example, may interact withcomplement components. N-glycans also serve to stabilize a glycoprotein,for example, by enhancing its solubility, shielding hydrophobic patcheson its surface, protecting from proteolysis, and directing intra-chainstabilizing interactions. Glycosylation may regulate protein half-life,for example, in humans the presence of terminal sialic acids inN-glycans may increase the half-life of proteins, circulating in theblood stream.

As used herein, the term “glycoprotein” refers to any protein having oneor more N-glycans attached thereto. Thus, the term refers both toproteins that are generally recognized in the art as a glycoprotein andto proteins which have been genetically engineered to contain one ormore N-linked glycosylation sites. As used herein, the terms “N-glycan”and “glycoform” are used interchangeably and refer to an N-linkedoligosaccharide, for example, one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-linked glycoproteins contain an N-acetylglucosamineresidue linked to the amide nitrogen of an asparagine residue in theprotein. The predominant sugars found on glycoproteins are glucose,galactose, mannose, fucose, N-acetylgalactosamine (GalNAc).N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminicacid (NANA)). The processing of the sugar groups occursco-translationally in the lumen of the ER and continuespost-translationally in the Golgi apparatus for N-linked glycoproteins.

A number of yeasts, for example, Pichia pastoris, Yarrowia lipolyticaand Saccharomyces cerevisiae are recently under development to use theadvantages of such systems but to eliminate the disadvantages in respectto glycosylation. Several strains are under genetical development toproduce defined, human-like glycan structures on a protein. Methods forgenetically engineering yeast to produce human-like N-glycans aredescribed in U.S. Pat. Nos. 7,029,872 and 7,449,308 along with methodsdescribed in U.S. Published Application Nos. 20040230042, 20050208617,20040171826, 20050208617, and 20060286637. These methods have been usedto construct recombinant yeast that can produce therapeuticglycoproteins that have predominantly human-like complex or hybridN-glycans thereon instead of yeast type N-glycans. As previouslydescribed, human-like glycosylation is primarily characterized by“complex” N-glycan structures containing N-acetylglucosamine, galactose,fucose and/or N-acetylneuraminic acid. Thus, several strains of yeastshave been genetically engineered to produce glycoproteins comprising oneor more human-like complex or human-like hybrid N-glycans such asGlcNAcMan3GlcNAc2.

As used herein, the term “skeletal growth retardation disorder” refersto a skeletal disease characterize by deformities and/or malformationsof the bones.

These disorders include, but are not limiting to, skeletal growthretardation disorders caused by growth plate (physeal) fractures,idiopathic skeletal growth retardation disorders and FGFR3-relatedskeletal diseases.

As used herein, the term “idiopathic skeletal growth retardationdisorder” refers to a skeletal disease whose the cause is unknown andfor which treatment with exogenous growth hormone (GH), e.g. recombinanthuman GH (rhGH), for instance has been shown to be ineffective.

In the context of the present invention, the term “FGFR3-relatedskeletal disease” is intended to mean a skeletal disease that is causedby an abnormal increased activation of FGFR3, in particular byexpression of a constitutively active mutant of the FGFR3 receptor Asused herein, the expressions “constitutively active FGFR3 receptorvariant”, “constitutively active mutant of the FGFR3” or “mutant FGFR3displaying a constitutive activity” are used interchangeably and referto a mutant of said receptor exhibiting a biological activity (i.e.triggering downstream signaling) in the absence of FGF ligandstimulation, and/or exhibiting a biological activity which is higherthan the biological activity of the corresponding wild-type receptor inthe presence of FGF ligand.

The FGFR3-related skeletal diseases are preferably FGFR3-relatedskeletal dysplasias and FGFR3-related craniosynostosis.

The FGFR3-related skeletal dysplasias according to the invention maycorrespond to an inherited or to a sporadic disease.

As used herein, the term “FGFR3-related skeletal dysplasias” includesbut is not limited to thanatophoric dysplasia type I, thanatophoricdysplasia type 1, hypochondroplasia, achondroplasia and SADDAN (severeachondroplasia with developmental delay and acanthosis nigricans).

In a preferred embodiment, the FGFR3-related skeletal dysplasia iscaused by expression in the subject of a constitutively active FGFR3receptor variant such as defined above.

In a preferred embodiment, the FGFR3-related skeletal dysplasia is anachondroplasia caused by expression of the G380R constitutively activemutant of the FGFR3 receptor.

In a preferred embodiment, the FGFR3-related skeletal dysplasia is ahypochondroplasia caused by expression of the N540K, K650N, K650Q, S84L,R200C, N262H, G268C, Y278C, S279C, V381E, constitutively active mutantof the FGFR3 receptor.

In a preferred embodiment, the FGFR3-related skeletal dysplasia is athanatophoric dysplasia type 1 caused by expression of a constitutivelyactive mutant of the FGFR3 receptor chosen from the group consisting ofR248C, S248C, G370C, S371C; Y373C, X807R, X807C, X807G, X807S, X807W andK650M FGFR3 receptors.

In a preferred embodiment, the FGFR3-related skeletal dysplasia is athanatophoric dysplasia type 11 caused by expression of the K650Econstitutively active mutant of the FGFR3 receptor.

In a preferred embodiment, the FGFR3-related skeletal dysplasia is asevere achondroplasia with developmental delay and acanthosis nigricanscaused by expression of the K650M constitutively active mutant of theFGFR3 receptor.

The present invention also provides a method for preventing or treatinga skeletal growth retardation disorder comprising the step ofadministering a therapeutically effective amount of a soluble FGFR3(sFGFR3) polypeptide to a subject in need thereof.

By a “therapeutically effective amount” of a sFGFR3 as above describedis meant a sufficient amount of the antagonist to prevent or treat aFGFR3-related skeletal disease (e.g. achondroplasia). It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of the specific compoundemployed; the specific composition employed, the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment: drugs used in combination orcoincidental with the specific polypeptide employed; and like factorswell known in the medical arts. For example, it is well within the skillof the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the subject to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 7 mg/kg of body weight per day.

As used herein, the term “subject” denotes a human or non-human mammal,such as a rodent, a feline, a canine, or a primate. Preferably, thesubject is a human being, more preferably a child (i.e. a child who isgrowing up).

In one embodiment, the subject has been diagnosed as suffering from aFGFR3-related skeletal disease. As previously described, theFGFR3-related skeletal disease is caused by expression in the subject ofa constitutively active FGFR3 receptor variant such as the G380Rconstitutively active mutant.

In the context of the invention, the term “treating” is used herein tocharacterize a therapeutic method or process that is aimed at (1)slowing down or stopping the progression, aggravation, or deteriorationof the symptoms of the disease state or condition to which such termapplies; (2) alleviating or bringing about ameliorations of the symptomsof the disease state or condition to which such term applies; and/or (3)reversing or curing the disease state or condition to which such termapplies.

As used herein, the term “preventing” intends characterizing aprophylactic method or process that is aimed at delaying or preventingthe onset of a disorder or condition to which such term applies.

Pharmaceutical Compositions of the Invention

The isolated soluble FGFR3 polypeptide (sFGFR3) as described above maybe combined with pharmaceutically acceptable excipients, and optionallysustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions.

Accordingly, the present invention also relates to a pharmaceuticalcomposition comprising an isolated sFGFR3 polypeptide according to theinvention and a pharmaceutically acceptable carrier.

The present invention further relates to a pharmaceutical compositionfor use in the prevention or treatment of a skeletal growth retardationdisorder comprising a sFGFR3 according to the invention and apharmaceutically acceptable carrier.

In one embodiment, the skeletal growth retardation disorder is anidiopathic growth retardation disorder.

In another embodiment, the skeletal growth retardation disorder is aFGFR3-related skeletal disease.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc. The pharmaceutical compositions of theinvention can be formulated for a topical, oral, intranasal,intraocular, intravenous, intramuscular or subcutaneous administrationand the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment. For example, it is well within the skillof the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the subject to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 7 mg/kg of body weight per day.

To prepare pharmaceutical compositions, an effective amount of apolypeptide according to the invention may be dissolved or dispersed ina pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The polypeptides according to the invention can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution may be suitably buffered and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. In this connection,sterile aqueous media which can be employed will be known to those ofskill in the art in light of the present disclosure. For example, onedosage could be dissolved in 1 ml of isotonic NaCl solution and eitheradded to 1000 ml of hypodermoclysis fluid or injected at the proposedsite of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the subject.

Another aspect of the present invention is a pharmaceutical compositionfor use in the prevention or treatment of a skeletal growth retardationdisorder comprising an isolated sFGFR3 polypeptide or a functionalequivalent thereof according to the invention and a pharmaceuticallyacceptable carrier.

In one embodiment, the skeletal growth retardation disorder is anidiopathic growth retardation disorder.

In another embodiment, the skeletal growth retardation disorder is aFGFR3-related skeletal disease.

The present invention also provides a method for preventing or treatinga skeletal growth retardation disorder comprising a step ofadministering a pharmaceutical composition comprising a therapeuticallyeffective amount of a sFGFR3 polypeptide or a functional equivalentthereof and a pharmaceutically acceptable carrier to a subject in needthereof.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1 : Effective FGF binding and decreased Erk phosphorylation inATDC5 cells in presence of FLAG-sFGFR3. (A) Fixed amounts of human ormurine basic FGF (100 ng) were incubated with increasing concentrationsof FLAG-sFGFR3. After 2 h, remaining unbound FGFs were detected byELISA. Lincar regression analysis showed no statistical differencesbetween the two slopes, hFGF, human FGFb; mFGF, mouse FGFb. Experimentwas performed in triplicate and repeated five times. (B) Erkphosphorylation was evaluated by immunoblotting on ATDC5 cells followingincubation with increasing doses of FLAG-sFGFR3. The graph representsthe phosphorylation variations in percentage compared to phosphorylationlevels in untreated cells. Experiments were repeated six times.Following verification of normality, statistical comparisons wereperformed using a one way ANOVA. *p<0.05, ***p<0.001. Values representmean±SD.

FIG. 2 : FLAG-sFGFR3 treatment effect on overall skeletal growth. (A)X-ray radiographies illustrating treatment effect on skeletal growth.Showed skeletons are representative of wt and Fgfr3^(ach/+) mice thatreceived subcutaneous injection of PBS or 5 ng FLAG-sFGFR3. Growth wascharacterized by body weight (B), body and tail lengths (C), and longbone measurements (D). (Data followed normal distribution; a Student's ttest was used to compare data to measurements obtained on untreatedmice. n per group are shown in Table 1; *p<0.05; **p<0.01; ***p<0.001versus untreated wt; ##p<0.0.1; ###p<0.001 versus untreatedFgfr3^(ach/+) mice. wt: wildtype mice; ach: Fgfr3^(ach/+) mice

FIG. 3 : Effect of FLAG-sFGFR3 treatment on vertebrae maturation. (A)The kyphosis index (KI) was measured from radiographs of mice positionedin right lateral recumbency. As defined by Laws et al. (28), line AB isthe length of a line drawn from posterior edge of C7 to the posterioredge of L6. Line CD is the distance from line AB to the dorsal border ofthe vertebral body farthest from that line. Clinically, a kyphosis ischaracterized with KI<4. (B) Photographs of representative vertebraefrom untreated wt, untreated Fgfr3^(ach/+) mice and transgenic micereceiving 5 ng FLAG-sFGFR3. In the table are indicated the percentage ofanimals in the different treatment groups with immature C7, T11 andlumbar vertebrae. ^(§) Lumbar compressions were characterized byparaplegia or locomotion deficiency. Data followed normal distribution;a Student's t test was used to compare data to measurements obtained onuntreated mice. n per group are shown in Table 1; *p<0.05; ***p<0.001versus untreated wt; ^(##)p<0.01; ^(###)p<0.001 versus untreatedFgfr3^(ach/+) mice, wt: wildtype mice; ach: Fgfr3^(ach/+) mice.

FIG. 4 : FLAG-sFGFR3 treatment effects on skull development. (A) Skulllength (L) and width (W) were measured and the ratio L/W calculated.Statistical analysis was performed using a Student's t test followingverification of normal variance and distribution. n per group are shownin Table 1; p<0.001 versus untreated wt; *p<0.05 versus untreatedFgfr3^(ach/+) mice. (B) Representative X-rays of skulls from wt andFgfr3^(ach/+) mice that received either PBS or 5 ng FLAG-sFGFR3. Theyshow treatment prevention of premature closure of cranial synchondrosetypically observed on Fgfr3^(ach/+) mice. This is indicated by thearrowhead. wt: wildtype mice; ach: Fgfr3^(ach/+) mice.

EXAMPLE

Material & Methods

sFGFR3 subcloning and recombinant protein production: To facilitatesub-cloning, full-length cDNA sequence encoding the FGFR3 ATM (2.1 kb)(35), a generous gift from Dr. Kurokawa-Seo, Kyoto Sangyo University,Japan, was optimized to decrease GC content while encoding for theoriginal protein sequence (GeneOptimizer® process, GeneArt). Thesynthesized fragment was subcloned into pFLAG-CMV3_G727 (Sigma Aldrich)using HindIII and KpnI cloning sites. Plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The final construct was verified by sequencing. The sequence homologywithin the used restriction sites was 100%.

Recombinant FLAG-sFGFR3 protein was produced by transient transfectionusing GeneJuice transfection reagent (Merck Millipore) in HEK 293 cellsallowing all necessary post-translational modifications. Eachtransfection was performed in a cell factory (High flask T600, MerckMillipore) with 80% confluent HEK 293 in 100 ml DMEM without phenol red(Gibco, Life Technologies) supplemented with glutamine 2 mM (Gibco, LifeTechnologies) and 1% antibiotics (Gibco, Life Technologies). 600 μlGeneJuice and 240 μg pFLAG-sFGFR3 were resuspended in 30 ml OptiMEM(Gibco, Life Technologies), incubated 30 min at room temperature, andthen incubated for 4 h onto the cells at 37° C. in 5% CO₂. Medium wasthen replaced by 120 ml DMEM without phenol red, supplemented withglutamine 2 mM and 1% antibiotics. After 72 h, production medium wasfiltrated using 0.22 μm filters and concentrated on Amicon Ultra-15 60kDa (Merck Millipore). Recombinant protein was then purified on anaffinity column (ANTI-FLAG M2 Affinity Gel. Sigma Aldrich) according tothe manufacturer's instructions. FLAG-sFGFR3 amounts were measured byspecific ELISA (R&D Systems) according to the manufacturer'sinstructions. FLAG-sFGFR3 was then stored at a concentration of 0.5μg/ml in 50% glycerol solution.

FLAG-sFGFR3 incubation with FGF: Fixed amounts of human or murine FGFb(100 μg) (R&D Systems) were incubated for 2 h at 37° C. with increasingdoses of FLAG-sFGFR3 (0 to 250 ng/ml) in PBS 1% BSA. Specific commercialELISA kits (R&D Systems) were used to quantify remammg unbound FGFs. Allexperiments were performed m triplicates and repeated five times.

Immunoblotting analysis: Immunoblotting was performed followingincubation of several doses of FLAG-sFGFR3 on ATDC5 cells. For this,ATDC5 cells were plated at a density of 2×10⁶ in 6 well plates and,following adhesion, cultured for 48 h in 0.5% BSA in DMEM-F12 (Gibco,Life Technologies) containing 1% antibiotics. Cells were then culturedfor 10 min with 100 μg/ml murine FGF pre-incubated for 2 hat 37° C. withincreasing doses of FLAG-sFGFR3 (0, 12.5, 125, 1250, 12500 μg/ml). Atthe end of the incubation period, remaining unbound FGFs were measuredby specific ELISA (R&D Systems). Cells were then solubilized in lysisbuffer (20 mM Tris. pH 7.4, 150 mM NaCl, 10 mM EDTA, 150 mM NaF, 2 mMsodium orthovanadate, 10 mM pyrophosphate, proteases inhibitors, and 1%Triton X-100) for 45 min at 4° C. Lysates were cleared (14 000 rpm, 10min) and proteins were separated by SDS-PAGE and immunoblotted aspreviously described (36). The proteins were probed with anti-phosphop42/44 MAPK (4370S, Cell Signaling), anti-total p42/44 MAPK (4696S, CellSignaling) and anti-hsp60 (scl 722, Santa Cruz Biotechnology) antibodies(1 μg/ml). All experiments were performed six times.

Immunohistochemistry of FLAG-sFGFR3: Immunohistochemistry of FLAG-sFGFR3was performed on tibiae of 3 day old Fgfdachl+ mice and their wildtypelittermates. For this, following decapitation of newborn mice, tibiaewere carefully harvested and incubated in 24 well plates in presence of5 ng FLAG-sFGFR3 for 24 hat 37° C. in 5% CO2. Tibiae were then rinsed inPBS and fixed in 10% formalin for 24 h. Following decalcification inEDTA for 2 days, bones were paraffin embedded and 5 μm sections wereincubated with 5 μg/ml anti-FLAG M2-FITC monoclonal antibody (SigmaAldrich). Sections were counterstained with Hoechst solution andvisualized under fluorescent microscopy. An anti-IgG antibody was usedas negative control.

Animals and treatments: The Principles of Laboratory Animal Care (NTHpublication no. 85-23, revised 1985;grantsl.nih.gov/grants/olaw/references/phspol.htm) and the Europeancommission guidelines for the protection of animals used for scientificpurposes(europa.eu/environment/chemicals/lab_animals/legislation_en.htm) werefollowed at all times. All procedures were approved by the InstitutionalEthic Committee for the use of Laboratory Animals (CIEPAL Azur)(approval #NCE-2012-52).

Experiments were performed on transgenic Fgfr3^(ach/+) animals in whichexpression of the mutant FGFR3 is driven by the Col2a1 promoter/enhancer(22). Mice were exposed to a 12 h light/dark cycle and had free accessto standard laboratory food and water. All measurements and analyseswere performed blinded and genotypes were analyzed after all analyseswere done by PCR of genomic DNA which amplify 360 bp of the FGFR3transgene (22). Two doses of FLAG-sFGFR3 (0.5 ng and 5 ng in 10 μl PBSwith 50% glycerol) were tested. At day 3, all newborn mice from a singlelitter received the same dose. Control litters received 10 μl of PBScontaining 50% glycerol. Subcutaneous injections were thereafter donetwice a week for three weeks, alternatively on the left and right sidesof the back. Mice were observed daily with particular attention tolocomotion and urination alterations. At day 22, all animals but twolitters per group were sacrificed by CO₂ asphyxation; genus andgenotypes were determined. Body weights were measured. Blood washarvested by cardiac puncture and mixed with 50 μl 0.5M EDTA; half ofthe samples were centrifuged for a biochemical assessment using aBeckman AU 2700 Analyzer (electrolytes (Na⁺, K⁺, Cl⁻), lactatedehydrogenase (LDH), cholesterol, creatinin, creatinin kinase (CK),aspartate aminotransferase (AST), alanine aminostransferase (ALT),amylase, total bilirubin (BLT)); the other half was analyzed withoutcentrifugation for blood numeration (Hemavet 950FS, Mascot Hematology).Cadavers were carefully skinned and eviscerated and skeletalmeasurements (body and tail lengths) were obtained using an electronicdigital caliper (Fisher Scientific). Total body length was measured fromthe nose to the end of the last caudal vertebra; tail was measuredstarting at the first caudal vertebra. Organs (heart, lungs, liver,kidneys, spleen) were harvested, weight and stored in 10% formalin forfurther histological analysis using standard paraffin-embeddedtechniques. X-rays of all skeletons were taken using a Faxitron X-raymachine (Edimex). Using an established method (28), kyphotic index weremeasured for each animals on the X-rays. Cleared skeletons were thenstained simultaneously with alcian blue and alizarin red using standardprocedures and stored in glycerol prior to analysis. Stained long bones(tibiae, femurs, humerus) were dissected and measured using anelectronic digital caliper; vertebrae and skulls were also dissected andanalyzed.

Breeding was set up to theoretically generate litters with half wildtypeand half heterozygous Fgfr3^(ach/+) mice. To avoid bias due tovariations of phenotype penetrance, experiments were performed on atleast 2 litters (one treated and one control) arising from the samebreeders. A total of 15, 9 and 11 litters representing a total of 312pups were treated with PBS, 0.5 ng or 5 ng FLAG-sFGFR3, respectively.The n per group is presented in Table 1.

Effect of FLAG-sFGFR3 on the fertility of treated animals: Animals fromthe litters that were not used for skeletal measurements were kept untilbreeding age was reached. At age 8 week, they were then mated with 8week old FVB/N mice from Charles River. Newborn mice were counted atbirth for each treated and control male and female and compared withfertility statistics of the previous generation. At age 22, offspringwere euthanized and growth was evaluated as described above.

Statistical analysis: All experiments and data measurements wereperformed by blind experimenters at all times. Statistical analyses wereperformed with GraphPad Prism 6.0 software. To determine the statisticaltests to be used, necessary assumptions were verified. To verifynormality and equal variance, an Agostino and Pearson omnibus normalitytest and a Brown-Forsythe test were performed, respectively. Because allskeletal measurements data sets fulfilled normality and equal variancerequirements, two-tailed Student's t test for comparisons of twoindependent groups were used in the different statistical analyses.Comparison of mortality data between treated and control groups was doneusing a Kruskal-Wallis test. Comparison of FLAG-sFGFR3 binding to humanand murine FGFs was done by linear regression. Immunoblotting datadistribution followed normality and were thus analyzed using a one-wayANOVA using a Holm-Sidak's multiple comparisons test. For organ weightcorrelation analyses, Pearson or Spearman tests were used when data setsfollowed or not normal distribution, respectively. All statistical testswere considered significant at a p<0.05 level of error. In all figures,values of p are shown as follows: *p<0.05; **p<0.01; ***p<0.001. Dataare presented as means±SD.

Results

FLAG-sFGFR3 effectively binds FGFs and decreases MAPK signaling in ATDC5cells: In order to detect recombinant soluble FGFR3 in vivo, theinventors used a soluble form of FGFR3 labeled with a FLAG tag. This tagwas used because of the availability of reagents for its purificationand its detection (23, 24). It has also already been used in vivowithout inducing premature elimination of the tagged protein by theimmune system (25, 26). In the present experiments, recombinantFLAG-sFGFR3 was produced by transient transfection, purified usingaffinity column and stored at a concentration of 0.5 μg/ml in 50%glycerol.

To verify that FLAG-sFGFR3 effectively bound free FGFs, fixed amounts ofhuman FGFb were incubated with increasing quantities of FLAG-sFGFR3. Asseen in FIG. 1A, FLAG-sFGFR3 effectively bound hFGF in a dose-dependentmanner. The FGFR3 ATM sequence used is of human origin, the inventorsverified that it could also bind murine FGF. Similar results wereobtained and FLAG-sFGFR3 was able to bind similar amounts of murineFGFs. This was expected since there is a 90% sequence homology betweenmurine and human FGFR3.

The inventors then verified that the complexation of FGF withFLAG-sFGFR3 resulted in a decreased intracellular FGF signaling on Erkphosphorylation. ATDC5 cells were used as a murine chondrocytic cellline to study chondrocyte biology (27). As seen FIG. 1B, significantdecrease in Erk phosphorylation was seen in relation with the dose ofFLAG-sFGFR3. This was correlated with a decrease of free FGFs in theconditioned medium, similar to that observed in FIG. 1A. These resultsdemonstrate that FLAG-sFGFR3 effectively binds FGFs of human and murineorigin thus decreasing FGF intracellular signaling.

Soluble FGFR3 effectively restores bone growth in Fgfr3^(ach/+) mice:Prior to test FLAG-sFGFR3 treatment effect in vivo, the inventorsverified that it could penetrate the dense cartilaginous matrix of thegrowth plate and reach target chondrocytes. Long bones isolated fromthree day old Fgfr3^(ach/+) mice and their wildtype (wt) littermateswere incubated for 24 h in presence of 5 ng FLAG-sFGFR3. As seen in FIG.2 , the recombinant protein was detected within the matrix, near thechondrocytes of the tibial growth plate of wt and Fgfr3^(ach/+) mice.

To evaluate the biological effects of FLAG-sFGFR3 treatment on skeletalbone growth in Fgfr3^(ach/+) mice, all newborn mice from one litterreceived the same treatment without knowing their phenotype. Theyreceived a subcutaneous injection of 0.5 or 5 ng FLAG-sFGFR3, or PBS incontrol groups, twice a week during 3 weeks. The first observation wasthe significant reduction in mortality in treated compared to untreatedlitters. At the end of the treatment period, control groups containedabout a third of transgenic animals alive while in both treated groups,there were approximately 50% wt mice and 50% Fgfr3^(ach/+) (Table 1).Moreover, in the control litters, 31% of animals died prior to the endof the experiments compared to 11.8% and 6.7% in the 0.5 ng and 5 ngFLAG-sFGFR3 treated litters, respectively. This reduction in group sizewas due to premature death or euthanasia of paraplegic animals. When itwas possible, autopsy was performed and confirmed death due torespiratory failure as seen by the presence of blood within the lungs.Two animals died consequently to bowel obstruction. All of these animalswere Fgfr3^(ach/+), a confirmed by genotyping. No wild-type animal diedprematurely. It is noteworthy to emphasize that in the control group,the majority of affected Fgfr3^(ach/+) mice died from respiratoryfailure, while in the 5 ng FLAG-sFGFR3 treatment group, they mostlysuffered from paraplegia and only few had respiratory distress (Table1). Altogether these data indicate that with treatment fewer animalsdied, and those who died had a less severe phenotype.

TABLE 1 Number of pups in the different treatment groups at day 3 andday 22. Litters were considered as single entities and all newborn micefrom the same cage received the same treatment. Dead and alive animalswere counted daily. Autopsy revealed death by respiratory failure andbowel occlusion for 2 animals. Animals with paraplegia were euthanizedupon discovery and recorded in the dead animal group. All dead animalswere Fgfr3^(ach/+). Statistical comparison versus control group was doneusing the Kruskal-Wallis test. Number of litters Number of pups % deadanimals before day 22 per group Day 3 Day 22 (cause of death) PBS 15 13291 31% (23 by respiratory failure, (wt: 67; ach: 24) 2 by bowelocclusion, 16 by paraplegia) 0.5 ng SFGFR3 9 76 67 11.8% ** (4 byrespiratory (wt: 31; ach: 36) failure, 5 by paraplegia) 5 ng sFGFR3 11104 97 6.7% ** (1 by respiratory failure, (wt: 47; ach: 50) 6 byparaplegia) ** p < 0.01. wt: wildtype mice; ach: Fgfr3^(ach/+) mice.

At day 22, time of weaning, animals were sacrificed and their growth wasevaluated. The inventors first confirmed that there was no statisticaldifference between males and females (Table S1) and regrouped them forall subsequent analyses. As illustrated in FIG. 2A, FLAG-sFGFR3treatment had an effect on overall skeletal growth. While Fgfr3^(ach/+)mice were in average 20% lighter than their wt littermates, animalstreated with FLAG-sFGFR3 displayed a dose dependent increase in theirbody weight, reaching up to 33% of the weight of untreated transgenicmice (FIG. 2B). A dose dependent treatment effect was also observed onthe weight of wt animals. As seen in FIG. 2C, treatment induced a dosedependent increase in body and tail lengths of both Fgfr3^(ach/+) and wtanimals. Treated transgenic mice had a stature that was notsignificantly different from that of untreated wt controls, reaching upto 10% of the lengths of untreated Fgfr3^(ach/+) animals at the highdose correcting the initial discrepancy between transgenic and wt mice.Similar results were obtained on long bone lengths. Humerus, femurs andtibiae from treated Fgfr3^(ach/+) mice were longer than those ofuntreated transgenic mice and were statistically identical to thelengths of wt bones (FIG. 21D). FLAG-sFGFR3 treatment also had a dosedependent effect on the growth of long bones from wt mice. Histologyconfirmed treatment effect on chondrocyte maturation. TreatedFgfr3^(ach/+) mice exhibited organized and hypertrophic chondrocytes intheir growth plates similarly to wt mice.

Altogether, these results show that following chronic subcutaneousadministration of FLAG-sFGFR3 to neonate Fgfr3^(ach/+) mice, normal bonegrowth was restored and that it was also effective on skeletal growth ofanimals that do not trigger an FGFR3 activating mutation.

TABLE S1 Statistical comparison of body measurements between male andfemale in the different treatment groups. Following 3 weeks oftreatments (PBS, 0.5 ng or 5 ng FLAG-sFGFR3), animals were sacrificed atage 22 days. Body weight, body length and tail length were measured.Genus and genotypes were determined. Following verification of normalityvariation in each data set, measurements were compared between males andfemales within the same treatment and genotype group. Data followednormal distribution; a Student's t test was used to compare data tomeasurements obtained on untreated mice. No statistical difference wasfound in any group. Body weight Body length Tail length male vs n (g)(mm) (mm) female PBS wt male 41 11.09 ± 1.26 132.43 ± 5.45 70.49 ± 3.65ns female 26 10.67 ± 1.99 134.04 ± 6.76 71.76 ± 4.60 ach male 13  9.18 ±1.82 120.56 ± 5.71 64.36 ± 2.52 ns female 11  8.14 ± 1.55 113.95 ± 8.2260.03 ± 3.79 0.5 ng wt male 12 12.31 ± 1.27 134.55 ± 7.98 73.94 ± 6.35ns sFGFR3 female 19 12.03 ± 1.18 134.59 ± 6.46 72.25 ± 4.51 ach male 1910.50 ± 1.19 126.78 ± 9.38 69.48 ± 6.64 ns female 17  9.34 ± 1.47 121.49 ± 10.01 65.33 ± 7.96 5 ng wt male 23 13.29 ± 1.48 141.47 ± 6.7774.76 ± 5.42 ns sFGFR3 female 24 12.13 ± 1.85 138.73 ± 8.21 74.01 ± 6.01ach male 13 11.41 ± 3.14  130.43 ± 13.11 69.15 ± 9.07 ns female 37 10.72± 2.19  127.59 ± 11.58 67.43 ± 7.95 ns = non significant. wt: wildtypemice; ach: Fgfr3^(ach/+) mice.

FLAG-sFGFR3 treatment decreases spinal and skull deformities associatedwith achondroplasia in Fgfr3^(ach/+) mice: in Fgfr3^(ach/+) mice, spinalabnormalities are recognized in particular by the presence of a kyphosisthat can be characterized by the calculation of a kyphotic index (KI).In this scoring system, established by Laws et al. (28), mice with aKI<4.0 present a kyphosis (for more details, please see legend of FIG. 3). In the present study, while no wt animals presented a spinaldeformity, 80% of untreated Fgfr3^(ach/+) mice displayed cervicalkyphosis with an average K1 of 3.46±0.65 (FIG. 3A). With FLAG-sFGFR3treatment, this percentage significantly decreased to 17% and 6% in the0.5 ng and 5 ng groups, respectively. To further characterize vertebralmaturation, the inventors analyzed ossification of C7 and T11. As seenFIG. 3B, on untreated Fgfr3^(ach/+) mice, the 7^(th) cervical and the11^(th) thoracic were not fused at the midline in 88.9% and 70.1%,respectively. Following treatment, maturation was restored as seen bythe decrease in the number of immature vertebrae. No wt animals in anygroups presented immature vertebrae.

Similar to achondroplasia patients that typically have enlarged heads,Fgfr3^(ach/+) mice suffer from skull deformities. While cranium width(W) is not statistically different between transgenic and wt mice(10.35±0.28 mm vs 10.17±0.32 mm, respectively), the length (L) issignificantly shorter in Fgfr3^(ach/+) mice (18.11±0.75 mm vs 20.05±0.51mm in wt mice, respectively). This leads to a L/W ratio equal to1.75±0.77 in untreated Fgfr3^(ach/+) mice and equal to 1.94±0.05 incontrol wt mice (FIG. 4A). FLAG-sFGFR3 treatment induced adose-dependent correction of the cranium length, and the L/W ratio wasnot significantly different from that of untreated wt at the highestdose of FLAG-sFGFR3. As seen in FIG. 5B, treatment also prevented thepremature closure of cranial synchondrose typically observed inFgfr3^(ach/+) mice.

Altogether, these results show that FLAG-sFGFR3 treatment is effectiveat preventing the development of skeleton deformities associated withachondroplasia.

No toxicological effects are detected in treated animals: Because theinventors are using a systemic approach to deliver recombinant solubleFGFR3, they paid a particular attention to possible unwanted sideeffects. They analyzed organs of all 255 animals, performed biochemicaland numeration blood tests and verified fertility of some treatedanimals (two litters per group) including normality of their offspring.

Potential treatment side effects were first evaluated on several organs(liver, lung, heart, spleen, kidneys) at the time of sacrifice. Organswere observed macroscopically, weighted and randomly analyzedmicroscopically by histology. None of the 255 animals that receivedchronic subcutaneous injections of FLAG-sFGFR3 or PBS presentedmacroscopic abnormalities. Histology was performed on randomly selectedorgans in all groups and data were analyzed blindly by ananatomopathologist. No signs of toxicity were observed on anyhistological slides. In all control groups, organ weights werecorrelated with the mouse body weight (Table 2). In the treated groups,organs increased with enhanced bone growth. As an example, the lungs ofuntreated Fgfr3^(ach/+) mice were 156.0±88.7 mg. They increased to172.5±67.5 mg in the 5 ng treatment group, reaching the weight of lungsin untreated wt mice (170.5±36.3 mg). Similar results were found for allorgans and this weight augmentation was statistically correlated withbody weight increase in all groups (Table 2). To evaluate organfunctions, they performed biochemical blood tests including electrolytestitration, liver, kidney and spleen enzymes assays. All tests proved tobe statistically identical between Fgfr3^(ach/+) and wt animals in thetreated and control groups (Table S2). Blood counts were also analyzedand similarly, no differences between blood formulations were noticedbetween treated and control groups (Table S3).

TABLE 2 Coefficient correlation (r) between organ and body weight in thedifferent treatment groups. Treatment Liver Heart Lung Spleen Kidney PBS0.892 *** 0.748 *** 0.857 *** 0.655 *** 0.877 *** 0.5 ng 0.883 **  0.777**  0.731 *  0.774 *  0.777 **  sFGFR3 5 ng 0.881 *** 0.794 *** 0.720*** 0.584 **  0.531 *  sFGFR3 PBS 0.941 *** 0.943 *** 0.886 **  0.726**  0.848 *** 0.5 ng 0.921 *** 0.758 *  0.709 **  0.650 *  0.828 ***sFGFR3 5 ng 0.957 *** 0.983 *** 0.885 *** 0.883 *** 0.850 *** sFGFR3Pearson or Spearman tests were used for statistical analysis oforgan/body weights correlations in each treatment group; * p < 0.05; **p < 0.01; *** p < 0.001. wt: wildtype mice; ach: Fgfr3^(ach/+) mice.

TABLE S2 Blood biochemical parameters were not modified by FLAG-sFGFR3treatment. To evaluate treatment toxicity, at time of sacrifice, plasmafrom the PBS and 5 ng FLAG-SFGFR3 groups were analyzed using a BeckmanAU 2700 Analyzer. Overall health was evaluated by measurements ofelectrolytes (Na⁺, K⁺, Cl⁻), lactate dehydrogenase (LDH) andcholesterol. Kidney function was assessed by creatinin and creatininkinase (CK) assays. Liver and pancreas functions were assessed byaspartate aminotransferase (AST), alanine aminostransferase (ALT), totalbilirubin (BLT) and amylase, respectively. Statistical comparisons wereperformed using a one way ANOVA. No statistical difference was found inany group. Na K Cl LDH Cholesterol Treatment (mmol/L) (mmol/L) (mmol/L)(UI/L) (mmol/L) wt PBS 810.8 ± 80.8 9.26 ± 0.51 75.20 ± 3.81 1672 ± 1240.99 ± 0.15 5 ng 818.3 ± 69.4 8.75 ± 0.59 72.83 ± 1.14 2059 ± 478 0.90 ±0.16 sFGFR3 ach PBS 709.7 ± 90.5 8.97 ± 0.98 81.79 ± 3.79 1675 ± 2281.06 ± 0.11 5 ng 706.2 ± 57.0 9.20 ± 0.77 76.33 ± 0.88 1738 ± 402 1.02 ±0.08 sFGFR3 Creatinine Total Creatinine kinase AST ALT Amylasebilirubine Treatment (μmol/L) (UI/L) (UI/L) (UI/L) (UI/L) (μmol/L) wtPBS  9.00 ± 1.26 1389 ± 679  155.1 ± 27   65.5 ± 16.5 138.1 ± 4.7  22.8± 1.7 5 ng 10.20 ± 0.95 655 ± 180 137.0 ± 24.6 42.3 ± 4.2  112.0 ± 8.0 20.8 ± 0.4 sFGFR3 ach PBS  9.18 ± 1.60 551 ± 127 140.1 ± 22.9 48.2 ±14.6 129.8 ± 21.4 21.8 ± 1.1 5 ng 12.33 ± 1.43 943 ± 261 196.4 ± 51.277.7 ± 14.5 192.0 ± 33.3 20.3 ± 0.6 SFGFR3 wt: wildtype mice; ach:Fgfr3^(ach/+) mice.

TABLE S3 Blood counts were not modified by FLAG-sFGFR3 treatment. Theeffects of FLAG-SFGFR3 treatment on blood counts were evaluated onplasma samples at the time of sacrifice. Analysis included hemoglobin(Hb), hematocrit (Ht), white blood cells (WBC), red blood cells (RBC)and platelets (PLT) counts. The percentages of the different leukocytepopulations were evaluated (NE: neutrophil; LY: lymphocyte, MO:monocyte, EO: eosinophil; BA: basophil). Statistical comparisons wereperformed using a one way ANOVA. No statistical difference was found inany group. Treatment Hb (g/dL) Ht (%) WBC (K/μl) RBC (K/μl) PLT (K/μl)wt PBS 8.11 ± 0.11 16.82 ± 1.72 4.31 ± 0.39 5.63 ± 0.02 471.1 ± 26.9 5ng 8.56 ± 0.06 23.21 ± 1.33 5.28 ± 0.54 5.81 ± 0.01 447.0 ± 32.2 sFGFR3ach PBS 8.32 ± 0.26  22.5 ± 4.74 3.37 ± 0.49 5.47 ± 0.18  472.0 ± 112.85 ng 8.25 ± 0.15 17.44 ± 1.12 5.86 ± 5.81 5.67 ± 0.04 620.0 ± 28.8sFGFR3 Treatment NE (%) LY (%) MO (%) EO (%) BA (%) wt PBS 30.76 ± 1.8653.56 ± 2.49 7.52 ± 0.59 5.89 ± 0.44 2.27 ± 0.27 5 ng 25.54 ± 2.37 56.90± 3.15 9.78 ± 0.67 5.75 ± 0.54 1.83 ± 0.24 sFGFR3 ach PBS 24.25 ± 6.0860.02 ± 8.86 6.56 ± 1.48 6.59 ± 1.72 2.56 ± 0.59 5 ng 29.25 ± 1.47 51.88± 2.39 10.16 ± 0.82  6.99 ± 1.20 1.85 ± 0.33 sFGFR3 wt: wildtype mice;ach: Fgfr3^(ach/+) mice.

To evaluate unwanted side effects on fecundity, after the three weektreatment, animals were weaned and were mated at age 8 weeks with wt FVBmales or females from Charles River. As seen in Table 3, all treatedanimals were fertile and their offspring were of normal size with anapproximate 50% wt/50% Fgfr3^(ach/+) descendants. While Fgfr3^(ach/+)females usually have a first litter slightly reduced in size compared tothat of wt females, it is interesting to note that primiparous treatedtransgenic females had litters that were identical in size to wtprimiparous females, confirming enlargement of their pelvis followingtreatment (Table 3).

TABLE 3 FLAG-sFGFR3 treatment did not affect fertility of the treatedmice. Pups from two litters per group were weaned after the three weektreatment. They were mated at age 8 weeks with wt animals from CharlesRiver. The number of pups of the first litters was counted for eachprimiparous female. At age 22 days, animals were euthanized. Their bodygrowth was evaluated as previously. A Student's t test was used tocompare data to measurements of untreated wt animals generated fromcontrol genitors. % of wt and Body weight Body length Litter size ach atday 22 (g) (mm) Control ach ♂ × wt ♀ 9.2 ± 0.9 74% wt, 26% wt, 10.88 ±wt, 133.24 ± genitors ach 1.62 6.11 wt ♂ × ach ♀    7.7 ± 0.7 *** ach,8.66 ± ach, 117.25 ± 1.68 *** 6.96 *** sFGFR3 treated ach ♂ × wt ♀ 10.2± 1.9  % wt, % ach treated treated wt ♂ × wt ♀ 9.7 ± 0.9 N/A wt ♂ ×treated ach ♀ 10.6 ± 1.5  % wt, % ach wt ♂ × treated wt ♀ 9.8 ± 1.9 N/A*** p < 0.001 versus untreated wt. N/A: not applicable, wt: wildtypemice; ach: Fgfr3^(ach/+) mice.

Altogether these experiments did not highlight any complications fromthe FLAG-sFGFR3 treatment itself neither on blood formulation, organfunction and development nor fertility of the treated animals,suggesting that the use of a soluble form of FGFR3 may be a viabletreatment approach for clinical applications.

DISCUSSION

The present study validates the proof of concept that a therapeuticstrategy based on the use of a soluble form of FGFR3 can preventabnormal bone growth in mice carrying the achondroplasia mutation.Treatment was administered twice a week by subcutaneous injections tothe animals throughout the growth period. Following this three weektreatment period, ensuing endochondral bone growth led to normal,harmonious stature. Importantly, these effects were dose-dependent; thedose of 0.5 ng FLAG-sFGFR3 was sufficient to induce body weight andlength that were identical to that of untreated wt mice and at the doseof 5 ng, treated dwarf mice were even heavier and had longer long bonesthan untreated wt animals. Foremost, the present results emphasize thenotion that the achondroplasia mutation requires ligand binding to beactivated. Indeed, it has been demonstrated that in the case of theG380R mutation, FGFR3 activation is ligand-dependent (12), but there isstill no clear consensus in the literature (29-31), suggesting not onebut multiple mechanisms leading to prolonged intracellular signaling.

While it was essential that long bone growth be restored for thetreatment to be effective, it was critical to also significantly impactthe onset of complications, due to skeletal deformities. For theinventors, this is indispensable if one wants to develop a treatment forthe clinic. Restoration of vertebra maturation and normal closure ofcranial synchondroses in treated Fgfr3^(ach/+) mice had numerous effectson these animals. The first consequence was the reduced mortality amongthe transgenic population. As stated in the result section, autopsyrevealed respiratory failures in the majority of the cases. Based on theanatomical characteristics of the skull and vertebrae of the dwarf mice,the inventors believe that these were consequent to brainstemcompression, similar to what can be observed in achondroplasia patients.

A second outcome of treatment effect was a shift in the penetrance ofthe phenotype. While measurements could appear as though treatmenteffects were not totally dose-dependent and more importantly not aseffective as could be expected based on body weights, the inventorsbelieve that at the highest dose, treatment saved the smallestFgfr3^(ach/+) mice from brainstem compression and respiratory failure.Untreated, these animals would not have survived past week 1 or 2. Inthis treatment group, the inventors hypothesize that these animals arethose that remain very small even though they have less severecomplications.

Despite the increasing number of reports studying the mechanismsunderlying achondroplasia and related skeletal dysplasia, only threestudies have been published showing therapeutic strategies effectivelytested in mice with chondrodysplasia. Xie et al. recently published areport that intermittent PTH treatment partially rescues bone growth inmice with achondroplasia (32). In their study. PTH was administeredsubcutaneously at the dose of 100 μg/kg body weight per day for 4 weeksafter birth. Although the mechanism by which PTH affects FGFR3intracellular signaling is not clearly established, bone growth waspartially rescued in treated transgenic mice; PTH treated transgenicmice were still smaller than their wt littermates. In this study, onlyvery little information was mentioned related to achondroplasiacomplications, except for the partial rescue of cranial synchondrose.The lethal phenotype of TDI mice was also rescued with chronic PTHtreatment of pregnant females. Another potential therapeutic antagonistof FGFR3 signaling is the C-natriuretic peptide (CNP) (33). In thispaper, the authors treated transgenic mice with achondroplasia bycontinuous intravenous infusion of synthetic CNP for 3 weeks starting atage 4 week. It is believe that CNP increases the width of the growthplate accelerating growth plate activity. The major obstacle for use inhuman is the very short half-life of CNP, estimated to be 2.6 min inplasma (34).

A study has been very recently published describing the use of a newFGFR3-binding peptide that rescues the lethal phenotype and partiallyrestores the structural distortion of growth plates in TDII mice,observed on 12 pups (19). In this study, effects on MAPK signaling andbone growth correction were only partial and daily administration wasrequired probably due to the short half-life of the peptide. Here, theeffects of SFGFR3 were of higher magnitude, with complete restoration ofnormal stature. The inventors believe that the half-life of the solubleform of FGFR3 containing IgG like domains is significantly prolonged.Indeed, only 6 injections (between birth and weaning) were necessary tocompletely restore bone growth in the 86 treated Fgfr3ach/+ mice. Theycan imagine that only a few injections would be necessary to treatachondroplasia children as out-patients, starting during the first yearuntil puberty. This would substantially impact occurrence of injectionside effects, typically found with daily injection regimens. The use ofa soluble recombinant protein also allows for rapid termination duringtreatment if safety issues are raised and at puberty when bone growthceases. If necessary, it is also possible to alternate between treatmentand resting periods. By preventing the complications, sFGFR3 treatmentwould avoid the necessity of surgical interventions and also reduce anystress due to hospitalization. Furthermore, our study did not reveal anytoxicological effect on blood or fertility and offspring of treatedanimals. Current studies are ongoing to evaluate if the three weeksFGFR3 treatment had an effect on the long-term health of the treatedmice. As of today, treated mice are 6 month old and no apparent sideeffects are visible and blood tests are normal.

In conclusion, the present study demonstrates the viability of targetingFGFR3 in the extracellular compartment as an effective treatment torestore growth plate maturation and induce normal bone growth inachondroplasia. The absence of unwanted side effects validates its useas a promising therapy for this and related chondrodysplasia caused byactivating mutation in FGF receptors. Furthermore, in the present study,the inventors also report a positive effect of sFGFR3 treatment on thegrowth of wt animals. This is of importance suggesting its possibleinterest for the treatment of idiopathic growth retardations, or toprevent severe complications in other rare diseases such ashypophosphatasia.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. A method for treating an FGFR3-relatedskeletal growth retardation disorder in a subject in need thereof, themethod comprising administering to the subject a soluble FibroblastGrowth Factor Receptor 3 (sFGFR3) polypeptide, wherein the sFGFR3polypeptide comprises the amino acid sequence of SEQ ID NO:
 1. 2. Themethod of claim 1, wherein the FGFR3-related skeletal growth retardationdisorder is thanatophoric dysplasia type I (TDI), thanatophoricdysplasia type II (TDII), severe achondroplasia with developmental delayand acanthosis nigricans (SADDAN), hypochondroplasia, achondroplasia,Muenke syndrome, or Crouzon syndrome with acanthosis nigricans.
 3. Themethod of claim 2, wherein the FGFR3-related skeletal growth retardationdisorder is achondroplasia.
 4. The method of claim 2, wherein theFGFR3-related skeletal growth retardation disorder is hypochondroplasia.5. The method of claim 1, wherein the subject is a human.
 6. The methodof claim 1, wherein the FGFR3-related skeletal growth retardationdisorder is caused by expression in the subject of an FGFR3 variant thatexhibits ligand-dependent overactivation.
 7. The method of claim 6,wherein the FGFR3 variant comprises an amino acid substitution of aglycine residue at position 380 of wild-type FGFR3 with an arginineresidue (G380R).
 8. The method of claim 1, wherein the subject isadministered 0.0002 mg/kg/day to 20 mg/kg/day of the sFGFR3 polypeptide.9. The method of claim 1, wherein the subject is administered 0.001mg/kg/day to 7 mg/kg/day of the sFGFR3 polypeptide.
 10. The method ofclaim 1, wherein the sFGFR3 polypeptide is administered subcutaneously.11. The method of claim 1, wherein the sFGFR3 polypeptide isadministered intravenously.